Background: The proper application of quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) for the relative quantification of a target gene in gene profiling studies requires reference genes to normalize sample variations. Stable housekeeping genes for this purpose have never been investigated in the liver tissue of patients with chronic hepatitis.
Material/methods: Expression profiles of six functionally distinct housekeeping genes (ACTB, CYCC, GUS, HPRT1, PMM1, POLR2L) were examined by RT-PCR in liver specimens from 12 individuals with chronic hepatitis C or B. Two software programs, geNorm and NormFinder, were used to assess the expression stability of the studied genes.
Results: Crossing-point values of the candidate reference genes were recorded between 22 and 28. In three groups of patients (all patients, HCV patients, HBV patients) both programs identified GUS as the most stably expressed housekeeping gene (stability values: 0.275-0.360 and 0.095-0.107 determined by geNorm and NormFinder, respectively), followed by PMM1 (0.275-0.360 and 0.168-0.227), and POLR2L (0.347-0.397 and 0.319-0.388).
Conclusions: The genes GUS and PMM1 are recommended for normalization purposes in gene expression studies of liver tissue from patients with chronic hepatitis C or B. Using these genes in combination will ensure very reliable results.